Single domain serum albumin binding protein

ABSTRACT

Disclosed herein are single domain serum albumins binding proteins with improved thermal stability, binding affinities, and robust aggregation profiles. Also described are multispecific binding proteins comprising a single domain serum albumin binding protein according to the instant disclosure. Pharmaceutical compositions comprising the binding proteins disclosed herein and methods of using such formulations are provided.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.62/339,682 filed May 20, 2016 which is incorporated by reference hereinin its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 18, 2017, isnamed 47517-703_201_SL.txt and is 22,218 bytes in size.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference,and as if set forth in their entireties.

BACKGROUND OF THE INVENTION

Albumin is the most abundant plasma protein, is highly soluble, verystable and has an extraordinarily long circulatory half-life. Albumincan be used in a variety of ways to increase the circulatory half-lifeof therapeutic molecules. The present disclosure provides a singledomain albumin binding protein which can be used for extending thehalf-life of therapeutic molecules.

SUMMARY OF THE INVENTION

Provided herein in one embodiment is a single domain serum albuminbinding protein, comprising complementarity determining regions CDR1,CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as setforth in GFX₁X₂X₃X₄FGMS (SEQ ID NO. 1), X₁ is threonine, arginine,lysine, serine, or proline, X₂ is phenylalanine or tyrosine, X₃ isserine, arginine or lysine, X₄ is serine, lysine, arginine, or alanine;(b) the amino acid sequence of CDR2 is as set forth inSISGSGX₅X₆TLYAX₇SX₈K (SEQ ID NO. 2), X₅ is serine, arginine, threonine,or alanine, X₆ is aspartic acid, histidine, valine, or threonine, X₇ isaspartic acid, histidine, arginine, or serine, X₈ is valine or leucine;and (c) the amino acid sequence of CDR3 is as set forth in GGSLX₉X₁₀(SEQ ID NO. 3), X₉ is serine, arginine, threonine, or lysine, and X₁₀ isarginine, lysine, valine, proline, or asparagine, wherein X₁, X₂, X₃,X₄, X₅, X₆, X₇, X₈, X₉, and X₁₀ are not simultaneously threonine,phenylalanine, serine, serine, serine, aspartic acid, aspartic acid,valine, serine, and arginine, respectively. In some embodiments, thesingle domain serum albumin binding protein comprises the followingformula: f1-r1-f2-r2-f3-r3-f4, wherein, r1 is SEQ ID NO. 1; r2 is SEQ IDNO. 2; and r3 is SEQ ID NO. 3; and wherein f₁, f₂, f₃ and f₄ areframework residues selected so that said protein is at least eightypercent identical to the amino acid sequence set forth in SEQ ID NO. 10.In some embodiments, the single domain serum albumin binding proteincomprises an amino acid sequence wherein r1 comprises SEQ ID NO. 14, SEQID NO. 15 or SEQ ID NO. 16. In some embodiments, the single domain serumalbumin binding protein comprises an amino acid sequence wherein r2comprises SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20,SEQ ID NO. 21, or SEQ ID NO. 22. In some embodiments, the single domainserum albumin binding protein comprises an amino acid sequence whereinr3 comprises SEQ ID NO. 23 or SEQ ID NO. 24. In some embodiments, thesingle domain serum albumin binding protein comprises an amino acidsequence wherein r1 comprises SEQ ID NO. 14. In some embodiments, thesingle domain serum albumin binding protein comprises an amino acidsequence wherein r1 comprises SEQ ID NO. 15, r2 comprises SEQ ID NO. 17,and r3 comprises SEQ ID NO. 23. In some embodiments, the single domainserum albumin binding protein comprises an amino acid sequence whereinr1 comprises SEQ ID NO. 16, and r3 comprises SEQ ID NO. 23. In someembodiments, the single domain serum albumin binding protein comprisesan amino acid sequence wherein r1 comprises SEQ ID NO. 15, and r2comprises SEQ ID NO. 18. In some embodiments, the single domain serumalbumin binding protein comprises an amino acid sequence wherein r1comprises SEQ ID NO. 14, and r3 comprises SEQ ID NO. 23. In someembodiments, the single domain serum albumin binding protein comprisesan amino acid sequence wherein r1 comprises SEQ ID NO. 15, r2 comprisesSEQ ID NO. 19 and r3 comprises SEQ ID NO. 24. In some embodiments, thesingle domain serum albumin binding protein comprises an amino acidsequence wherein r1 comprises SEQ ID NO. 14, and r2 comprises SEQ ID NO.20. In some embodiments, the single domain serum albumin binding proteincomprises an amino acid sequence wherein r1 comprises SEQ ID NO. 15, andr2 comprises SEQ ID NO. 21. In some embodiments, the single domain serumalbumin binding protein comprises an amino acid sequence wherein r1comprises SEQ ID NO. 15, r2 comprises SEQ ID NO. 22, and r3 comprisesSEQ ID NO. 24. In some embodiments, the single domain serum albuminbinding protein has an amino acid sequence selected from SEQ ID NO. 4,SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9,SEQ IN NO. 25, SEQ ID NO. 26, and SEQ ID NO. 27. In some embodiments,the single domain serum albumin binding protein comprises the amino acidsequence set forth as SEQ ID NO. 4. In some embodiments, the singledomain serum albumin binding protein comprises the amino acid sequenceset forth as SEQ ID NO. 7. In some embodiments, the single domain serumalbumin binding protein comprises the amino acid sequence set forth asSEQ ID NO. 9. In some embodiments, the single domain serum albuminbinding protein comprises the amino acid sequence set forth as SEQ IDNO. 26. In some embodiments, the single domain serum albumin bindingprotein comprises the amino acid sequence set forth as SEQ ID NO. 27.

In some embodiments, the single domain serum albumin binding proteinbinds to serum albumin selected from human serum albumin, cynomolgusserum albumin, and mouse serum albumin. In some embodiments, the singledomain serum albumin binding protein binds to human serum albumin andcynomolgus serum albumin with comparable binding affinity (Kd). In someembodiments, the single domain serum albumin binding protein binds tomouse serum albumin with a binding affinity (Kd) that is about 1.5 foldto about 20 fold weaker than the binding affinity (Kd) of said proteintowards human and cynomolgus serum albumin. In some embodiments, thesingle domain serum albumin binding protein binds to human serum albuminwith a human Kd (hKd) between about 1 nM and about 100 nM and tocynomolgus serum albumin with a cynomolgus Kd (cKd) between 1 nM and 100nM. In some embodiments, the hKd and the cKd of the single domain serumalbumin binding protein are between 1 nM and about 5 nM, or about 5 nMand about 10 nM. In some embodiments, the hKd and cKd of the singledomain serum albumin binding protein are between about 1 nM and about 2nM, about 2 nM and about 3 nM, about 3 nM and about 4 nM, about 4 nM andabout 5 nM, about 5 nM and about 6 nM, about 6 nM and about 7 nM, about7 nM and about 8 nM, about 8 nM and about 9 nM, or about 9 nM and about10 nM. In some embodiments, the ratio between the hKd and cKd (hKd:cKd)of the single domain serum albumin binding protein ranges from about20:1 to about 1:2.

In some embodiments, the single domain serum albumin binding proteincomprises the amino acid sequence set forth as SEQ ID NO. 4, and whereinthe hKd and the cKd are between about 1 nM and about 5 nM. In someembodiments, the single domain serum albumin binding protein comprisesthe amino acid sequence set forth as SEQ ID NO. 5, and wherein the hKdand the cKd are between about 1 nM and about 5 nM. In some embodiments,the single domain serum albumin binding protein comprises the amino acidsequence set forth as SEQ ID NO. 6, and wherein the hKd and the cKd arebetween about 1 nM and about 5 nM. In some embodiments, the singledomain serum albumin binding protein comprises the amino acid sequenceset forth as SEQ ID NO. 7, and wherein the hKd and the cKd are betweenabout 1 nM and about 5 nM. In some embodiments, the single domain serumalbumin binding protein comprises the amino acid sequence set forth asSEQ ID NO. 8, and wherein the hKd and the cKd are between about 5 nM andabout 10 nM. In some embodiments, the single domain serum albuminbinding protein comprises the amino acid sequence set forth as SEQ IDNO. 9, and wherein the hKd and the cKd are between about 1 nM and about5 nM. In some embodiments, the single domain serum albumin bindingprotein comprises the amino acid sequence set forth as SEQ ID NO. 22,and wherein the hKd and the cKd are between about 1 nM and about 5 nM.In some embodiments, the single domain serum albumin binding proteincomprises the amino acid sequence set forth as SEQ ID NO. 23, andwherein the hKd and the cKd are between about 1 nM and about 5 nM. Insome embodiments, the single domain serum albumin binding proteincomprises the amino acid sequence set forth as SEQ ID NO. 24, andwherein the hKd and the cKd are between about 1 nM and about 5 nM.

In some embodiments, the single domain serum albumin binding proteincomprises elimination half-time of at least 12 hours, at least 20 hours,at least 25 hours, at least 30 hours, at least 35 hours, at least 40hours, at least 45 hours, at least 50 hours, or at least 100 hours.

In another embodiment is provided a single domain serum albumin bindingprotein comprising CDR1, CDR2, and CDR3, comprising the sequence setforth as SEQ ID NO. 10 wherein one or more amino acid residues selectedfrom amino acid positions 28, 29, 30, or 31 of CDR1; positions 56, 57,62, or 64 of CDR2; and positions 103, and 104 of CDR3 are substituted,wherein amino acid position 28 is substituted with arginine, lysine,serine, or proline, amino acid position 29 is substituted with tyrosine,amino acid position 30 is substituted with arginine or lysine, aminoacid position 31 is substituted with lysine, arginine, or alanine, aminoacid position 56 is substituted with arginine, threonine, or alanine,amino acid position 57 is substituted with histidine, valine, orthreonine, amino acid position 62 is substituted with histidine,arginine, glutamic acid, or serine, amino acid position 64 issubstituted with leucine, amino acid position 103 is substituted witharginine, threonine, or lysine, amino acid position 104 is substitutedwith lysine, valine, proline, or asparagine. In some embodiments, thesingle domain serum albumin binding comprises one or more additionalsubstitutions in amino acid positions other than positions 28, 29, 30,31, 56, 57, 62, 64, 103, and 104. In some embodiments, the single domainserum albumin binding protein comprises a substitution in position 29.In some embodiments, the single domain serum albumin binding proteincomprises a substitution in position 31. In some embodiments, the singledomain serum albumin binding protein comprises a substitution inposition 56. In some embodiments, the single domain serum albuminbinding protein comprises a substitution in position 62. In someembodiments, the single domain serum albumin binding protein comprises asubstitution in position 64. In some embodiments, the single domainserum albumin binding protein comprises a substitution in position 104.In some embodiments, the single domain serum albumin binding proteincomprises substitutions in amino acid positions 31 and 62. In someembodiments, the single domain serum albumin binding protein comprises aamino acid sequence wherein position 31 is substituted with arginine. Insome embodiments, the single domain serum albumin binding proteincomprises an amino acid sequence wherein position 31 is substituted witharginine and amino acid position 62 is substituted with glutamic acid.In some embodiments, the single domain serum albumin binding proteincomprises substitutions in amino acid positions 31, 56, 64, and 104. Insome embodiments, the single domain serum albumin binding proteincomprises an amino acid sequence wherein position 31 is substituted withlysine, amino acid position 56 is substituted with alanine, amino acidposition 64 is substituted with leucine, and amino acid position 104 issubstituted with lysine. In some embodiments, the single domain serumalbumin binding protein comprises substitutions in amino acid positions29 and 104. In some embodiments, the single domain serum albumin bindingprotein comprises an amino acid sequence wherein amino acid position 29is substituted with tyrosine, and amino acid position 104 is substitutedwith lysine. In some embodiments, the single domain serum albuminbinding protein comprises substitutions in amino acid positions 31 and56. In some embodiments, the single domain serum albumin binding proteincomprises an amino acid sequence wherein amino acid position 31 issubstituted with lysine, and amino acid position 56 is substituted withthreonine. In some embodiments, the single domain serum albumin bindingprotein comprises substitutions in amino acid positions 31, 56, and 62.In some embodiments, the single domain serum albumin binding proteincomprises an amino acid sequence wherein amino acid position 31 issubstituted with lysine, amino acid position 56 is substituted withthreonine, and amino acid position 62 is substituted with glutamic acid.In some embodiments, the single domain serum albumin binding proteincomprises substitutions in amino acid positions 31 and 104. In someembodiments, the single domain serum albumin binding protein comprisesan amino acid sequence wherein amino acid position 31 is substitutedwith arginine, and amino acid position 104 is substituted with lysine.In some embodiments, the single domain serum albumin binding proteincomprises substitutions in amino acid positions 31, 56, and 104. In someembodiments, the single domain serum albumin binding protein comprisesan amino acid sequence wherein amino acid position 31 is substitutedwith lysine, amino acid position 56 is substituted with arginine, andamino acid position 104 is substituted with valine. In some embodiments,the single domain serum albumin binding protein comprises substitutionsin amino acid positions 31, 56, 62, and 104. In some embodiments, thesingle domain serum albumin binding protein comprises an amino acidsequence wherein amino acid position 31 is substituted with lysine,amino acid position 56 is substituted with arginine, amino acid position62 is substituted with glutamic acid, and amino acid position 104 issubstituted with valine. In some embodiments, the single domain serumalbumin binding protein comprises an amino acid sequence wherein aminoacid position 31 is substituted with arginine, and the hKd and the cKdof the single domain serum albumin binding protein are between about 1nM and about 5 nM. In some embodiments, the single domain serum albuminbinding protein comprises an amino acid sequence wherein amino acidposition 31 is substituted with arginine, amino acid position 62 issubstituted with glutamic acid, and the hKd and the cKd of the singledomain serum albumin binding protein are between about 1 nM and about 5nM. In some embodiments, the single domain serum albumin binding proteincomprises an amino acid sequence wherein amino acid position 31 issubstituted with lysine, amino acid position 56 is substituted withalanine, amino acid position 64 is substituted with leucine, amino acidposition 104 is substituted with lysine, and wherein the hKd and the cKdof the single domain serum albumin binding protein are between about 1nM and about 5 nM. In some embodiments, the single domain serum albuminbinding protein comprises an amino acid sequence wherein amino acidposition 29 is substituted with tyrosine, amino acid position 104 issubstituted with lysine, and wherein the hKd and the cKd are betweenabout 1 nM and about 5 nM. In some embodiments, the single domain serumalbumin binding protein comprises an amino acid sequence wherein aminoacid position 31 is substituted with lysine, amino acid position 56 issubstituted with threonine, and wherein the hKd and the cKd of thesingle domain serum albumin binding protein are between about 1 nM andabout 5 nM. In some embodiments, the single domain serum albumincomprises an amino acid sequence wherein amino acid position 31 issubstituted with lysine, amino acid position 56 is substituted withthreonine, and amino acid position 62 is substituted with glutamic acid,and wherein the hKd and the cKd are between about 1 nM and about 5 nM.In some embodiments, the single domain serum albumin binding proteincomprises an amino acid sequence wherein amino acid position 31 issubstituted with arginine, amino acid position 104 is substituted withlysine, and wherein the hKd and the cKd of the single domain serumalbumin binding protein are between about 5 nM and about 10 nM. In someembodiments, the single domain serum albumin binding protein comprisesan amino acid sequence wherein amino acid position 31 is substitutedwith lysine, amino acid position 56 is substituted with arginine, aminoacid position 104 is substituted with valine, and wherein the hKd andthe cKd of the single domain serum albumin binding protein are betweenabout 1 nM and about 5 nM. In some embodiments, the single domain serumalbumin binding protein comprises an amino acid sequence wherein aminoacid position 31 is substituted with lysine, amino acid position 56 issubstituted with arginine, amino acid position 62 is substituted withglutamic acid, and amino acid position 104 is substituted with valine,and wherein the hKd and the cKd of the single domain serum albuminbinding protein are between about 1 nM and about 5 nM.

Provided herein in another embodiment is a single domain serum albuminbinding protein comprising at least one mutation in CDR1, CDR2 or CDR3,wherein CDR1 comprises the sequence as set forth is SEQ ID NO:11, CDR2comprises the sequence as set forth in SEQ ID NO:12, CDR3 comprises thesequence as set forth in SEQ ID NO. 13, and wherein the at least onemutation is not in amino acid positions 1, 2, 7, 8, 9, or 10 of SEQ IDNO: 11, positions 1, 3, 6, 10, or 11 of SEQ ID NO: 12, or positions 1 or2 of SEQ ID NO: 13. In some embodiments, the single domain serum albuminbinding protein comprises at least one mutation in amino acid positionsselected from positions 3, 4, 5, and 6 of CDR1 (SEQ ID NO: 11), aminoacid positions 7, 8, 13, and 15 of CDR2 (SEQ ID NO: 12), and amino acidpositions 5 and 6 of CDR3 (SEQ ID NO: 13). In some embodiments, thesingle domain serum albumin binding protein comprises one or moreadditional substitutions in amino acid positions other than 3, 4, 5, and6 of CDR1 (SEQ ID NO: 11), amino acid positions 7, 8, 13, and 15 of CDR2(SEQ ID NO: 12), and amino acid positions 5 and 6 of CDR3 (SEQ ID NO:13). In some embodiments, the single domain serum albumin bindingprotein comprises a mutation in amino acid position 6 of CDR 1 (SEQ IDNO:11). In some embodiments, the single domain serum albumin bindingprotein comprises a mutation in amino acid position 6 of CDR 1 (SEQ IDNO:11), and amino acid position 13 of CDR2 (SEQ ID NO: 12). In someembodiments, the single domain serum albumin binding protein comprisesmutations in amino acid position 6 of (SEQ ID NO:11), amino acidpositions 7 and 15 of CDR2 (SEQ ID NO: 12), and amino acid position 6 ofCDR3 (SEQ ID NO: 13). In some embodiments, the single domain serumalbumin binding protein comprises mutations in amino acid position 4 ofCDR1 (SEQ ID NO:11), and amino acid position 6 of CDR3 (SEQ ID NO: 13).In some embodiments, the single domain serum albumin binding proteincomprises mutations in amino acid position 6 of CDR 1 (SEQ ID NO:11),and amino acid position 7 of CDR2 (SEQ ID NO: 12). In some embodiments,the single domain serum albumin binding protein comprises mutations inamino acid position 6 of CDR 1 (SEQ ID NO:11), and amino acid positions7 and 13 of CDR2 (SEQ ID NO: 12). In some embodiments, the single domainserum albumin binding protein comprises mutations in amino acid position6 of CDR1 (SEQ ID NO:11), and amino acid position 6 of CDR3 (SEQ ID NO:13). In some embodiments, the single domain serum albumin bindingprotein comprises mutations in amino acid position 6 of CDR1 (SEQ IDNO:11), amino acid position 7 of CDR2 (SEQ ID NO: 12), and amino acidposition 6 of CDR3 (SEQ ID NO: 13). In some embodiments, the singledomain serum albumin binding protein comprises mutations in amino acidposition 6 of CDR1 (SEQ ID NO:11), amino acid positions 7 and 13 of CDR2(SEQ ID NO: 12), and amino acid position 6 of CDR3 (SEQ ID NO: 13). Insome embodiments, the single domain serum albumin binding proteincomprises an amino acid sequence wherein amino acid position 6 of CDR1(SEQ ID NO:11) is mutated to arginine, and wherein the hKd and the cKdof the single domain serum albumin binding protein are between about 1nM and about 5 nM. In some embodiments, the single domain serum albuminbinding protein comprises an amino acid sequence wherein amino acidposition 6 of CDR1 (SEQ ID NO:11) is mutated to arginine, and amino acidposition 13 of CDR2 (SEQ ID NO: 12) is mutated to glutamic acid, andwherein the hKd and the cKd of the single domain serum albumin bindingprotein are between about 1 nM and about 5 nM. In some embodiments, thesingle domain serum albumin binding protein comprises an amino acidsequence wherein amino acid position 6 is of CDR 1 (SEQ ID NO:11) ismutated to lysine, amino acid positions 7 and 15 of CDR2 (SEQ ID NO: 12)are mutated to alanine and leucine, respectively, amino acid position 6of CDR3 (SEQ ID NO: 13) is mutated to lysine, and wherein the hKd andthe cKd of the single domain serum albumin binding protein are betweenabout 1 nM and about 5 nM. In some embodiments, the single domain serumalbumin binding protein comprises an amino acid sequence wherein aminoacid position 4 of CDR1 (SEQ ID NO:11) is mutated to tyrosine, and aminoacid position 6 of CDR3 (SEQ ID NO: 13) is mutated to lysine, andwherein the hKd and the cKd of the single domain serum albumin bindingprotein are between about 1 nM and about 5 nM. In some embodiments, thesingle domain serum albumin binding protein comprises an amino acidsequence wherein amino acid position 6 of CDR 1 (SEQ ID NO:11) ismutated to lysine, amino acid position 7 of CDR2 (SEQ ID NO: 12) ismutated to threonine, and wherein the hKd and the cKd of the singledomain serum albumin binding protein are between about 1 nM and about 5nM. In some embodiments, the single domain serum albumin binding proteincomprises an amino acid sequence wherein amino acid position 6 of CDR 1(SEQ ID NO:11) is mutated to lysine, amino acid positions 7 and 13 ofCDR2 (SEQ ID NO: 12) are mutated to threonine and glutamic acid,respectively, and wherein the hKd and the cKd of the single domain serumalbumin binding protein are between about 1 nM and about 5 nM. In someembodiments, the single domain serum albumin binding protein comprisesan amino acid sequence wherein amino acid position 6 of CDR1 (SEQ IDNO:11) is mutated to arginine, amino acid position 6 of CDR3 (SEQ ID NO:12) is mutated to lysine, and wherein the hKd and the cKd of the singledomain serum albumin binding protein are between about 5 nM and about 10nM. In some embodiments, the single domain serum albumin binding proteincomprises an amino acid sequence wherein amino acid position 6 of CDR 1(SEQ ID NO:11) is mutated to lysine, amino acid position 7 of CDR2 (SEQID NO: 12) is mutated to arginine, amino acid position 6 of CDR3 (SEQ IDNO: 13) is mutated to valine, and wherein the hKd and the cKd of thesingle domain serum albumin binding protein are between about 1 nM andabout 5 nM. In some embodiments, the single domain serum albumin bindingprotein comprises an amino acid sequence wherein amino acid position 6of CDR 1 (SEQ ID NO:11) is mutated to lysine, amino acid positions 7 and13 of CDR2 (SEQ ID NO: 12) is mutated to arginine and glutamic acid,respectively, and amino acid position 6 of CDR3 (SEQ ID NO: 13) ismutated to valine, and wherein the hKd and the cKd of the single domainserum albumin binding protein are between about 1 nM and about 5 nM.

Provided herein in another embodiment, a polynucleotide encoding asingle domain serum albumin binding protein according to the presentdisclosure. A further embodiment describes a vector comprising thepolynucleotide as disclosed herein. Another embodiment describes a hostcell transformed with the vector according to the present disclosure. Inone embodiment is provided a pharmaceutical composition comprising (i) asingle domain serum albumin binding protein according to the presentdisclosure, a polynucleotide according to the present disclosure, avector according to the present disclosure or a host cell according tothe present disclosure, and (ii) a pharmaceutically acceptable carrier.

Described herein in another embodiment, is a process for the productionof a single domain serum albumin binding protein according to thepresent disclosure, said process comprising culturing a host transformedor transfected with a vector comprising a nucleic acid sequence encodinga single domain serum albumin binding protein as described herein underconditions allowing the expression of the serum albumin binding proteinand recovering and purifying the produced protein from the culture.

Further described is a method for the treatment or amelioration of aproliferative disease, a tumorous disease, an inflammatory disease, animmunological disorder, an autoimmune disease, an infectious disease, aviral disease, an allergic reaction, a parasitic reaction, agraft-versus-host disease or a host-versus-graft disease comprising theadministration of the single domain serum albumin binding proteinaccording to the present disclosure, to a subject in need thereof. Insome embodiments, the subject is human. In some embodiments, the methodfurther comprises administration of an agent in combination with thesingle domain serum albumin binding protein according to the presentdisclosure.

In another embodiment is described a multispecific binding proteincomprising the single domain serum albumin binding protein according tothe present disclosure. In another embodiment is described an antibodycomprising the single domain serum albumin binding protein according tothe present disclosure.

A further embodiment describes a multispecific antibody, a bispecificantibody, an sdAb, a variable heavy domain, a peptide, or a ligand,comprising the single domain serum albumin binding protein according tothe present disclosure. In one embodiment is provided an antibodycomprising the single domain serum albumin binding protein according tothe present disclosure, wherein said antibody is a single domainantibody. In some embodiments, the single domain antibody is derivedfrom a heavy chain variable region of IgG.

One embodiment describes a multispecific binding protein or antibodycomprising the single domain serum albumin binding protein according tothe present disclosure and a CD3 binding domain. In one embodiment isdescribed a method for the treatment or amelioration of a proliferativedisease, a tumorous disease, an inflammatory disease, an immunologicaldisorder, an autoimmune disease, an infectious disease, a viral disease,an allergic reaction, a parasitic reaction, a graft-versus-host diseaseor a host-versus-graft disease comprising administration of themultispecific antibody according to the present disclosure, to a subjectin need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates the specific binding of parental anti-HSA phage asdetermined by ELISA titration to an HSA antigen and a CD3 antigen.

FIG. 2 illustrates the cross reactivity of anti-HSA phage to human,cynomolgus monkey, and mouse serum albumin as determined by ELISAtitration.

FIG. 3 provides the binding affinity profiles of nine clones selectedfor more precise Kd determinations using purified sdAbs.

FIG. 4 illustrates the temperature of hydrophobic exposure (T_(h)° C.)for several anti-HSA sdAb variants.

FIG. 5 illustrates the propensity of several anti-HSA sdAb variants toform dimer versus monomer at low pH.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby

Certain Definitions

The terminology used herein is for the purpose of describing particularcases only and is not intended to be limiting. As used herein, thesingular forms “a”, “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.Furthermore, to the extent that the terms “including”, “includes”,“having”, “has”, “with”, or variants thereof are used in either thedetailed description and/or the claims, such terms are intended to beinclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the given value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” should be assumed to mean an acceptable error range for theparticular value.

The terms “individual,” “patient,” or “subject” are usedinterchangeably. None of the terms require or are limited to situationcharacterized by the supervision (e.g. constant or intermittent) of ahealth care worker (e.g. a doctor, a registered nurse, a nursepractitioner, a physician's assistant, an orderly, or a hospice worker).

The term “Framework” or “FR” residues (or regions) refer to variabledomain residues other than the CDR or hypervariable region residues asherein defined. A “human consensus framework” is a framework whichrepresents the most commonly occurring amino acid residue in a selectionof human immunoglobulin VL or VH framework sequences.

As used herein, “Variable region” or “variable domain” refers to thefact that certain portions of the variable domains differ extensively insequence among antibodies and are used in the binding and specificity ofeach particular antibody for its particular antigen. However, thevariability is not evenly distributed throughout the variable domains ofantibodies. It is concentrated in three segments calledcomplementarity-determining regions (CDRs) or hypervariable regions bothin the light-chain and the heavy-chain variable domains. The more highlyconserved portions of variable domains are called the framework (FR).The variable domains of native heavy and light chains each comprise fourFR regions, largely adopting a β-sheet configuration, connected by threeCDRs, which form loops connecting, and in some cases forming part of,the βsheet structure. The CDRs in each chain are held together in closeproximity by the FR regions and, with the CDRs from the other chain,contribute to the formation of the antigen-binding site of antibodies(see Kabat et al., Sequences of Proteins of Immunological Interest,Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). Theconstant domains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody in antibody-dependent cellular toxicity. “Variabledomain residue numbering as in Kabat” or “amino acid position numberingas in Kabat,” and variations thereof, refers to the numbering systemused for heavy chain variable domains or light chain variable domains ofthe compilation of antibodies in Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991). Using this numbering system,the actual linear amino acid sequence may contain fewer or additionalamino acids corresponding to a shortening of, or insertion into, a FR orCDR of the variable domain. For example, a heavy chain variable domainmay include a single amino acid insert (residue 52 a according to Kabat)after residue 52 of H2 and inserted residues (e.g. residues 82 a, 82 b,and 82 c, etc according to Kabat) after heavy chain FR residue 82. TheKabat numbering of residues may be determined for a given antibody byalignment at regions of homology of the sequence of the antibody with a“standard” Kabat numbered sequence. It is not intended that CDRs of thepresent disclosure necessarily correspond to the Kabat numberingconvention.

As used herein, the term “Percent (%) amino acid sequence identity” withrespect to a sequence is defined as the percentage of amino acidresidues in a candidate sequence that are identical with the amino acidresidues in the specific sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

As used herein, “elimination half-time” is used in its ordinary sense,as is described in Goodman and Gillman's The Pharmaceutical Basis ofTherapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and AlfredGilman, eds., 6th ed. 1980). Briefly, the term is meant to encompass aquantitative measure of the time course of drug elimination. Theelimination of most drugs is exponential (i.e., follows first-orderkinetics), since drug concentrations usually do not approach thoserequired for saturation of the elimination process. The rate of anexponential process may be expressed by its rate constant, k, whichexpresses the fractional change per unit of time, or by its half-time,t_(1/2) the time required for 50% completion of the process. The unitsof these two constants are time⁻¹ and time, respectively. A first-orderrate constant and the half-time of the reaction are simply related(k×t_(1/2)=0.693) and may be interchanged accordingly. Since first-orderelimination kinetics dictates that a constant fraction of drug is lostper unit time, a plot of the log of drug concentration versus time islinear at all times following the initial distribution phase (i.e. afterdrug absorption and distribution are complete). The half-time for drugelimination can be accurately determined from such a graph.

As used herein, the term “binding affinity” refers to the affinity ofthe proteins described in the disclosure to their binding targets, andis expressed numerically using “Kd” values. If two or more proteins areindicated to have comparable binding affinities towards their bindingtargets, then the Kd values for binding of the respective proteinstowards their binding targets, are within ±2-fold of each other. If twoor more proteins are indicated to have comparable binding affinitiestowards single binding target, then the Kd values for binding of therespective proteins towards said single binding target, are within±2-fold of each other. If a protein is indicated to bind two or moretargets with comparable binding affinities, then the Kd values forbinding of said protein to the two or more targets are within ±2-fold ofeach other. In general, a higher Kd value corresponds to a weakerbinding. In some embodiments, the “Kd” is measured by a radiolabeledantigen binding assay (MA) or surface plasmon resonance assays using aBIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.). Incertain embodiments, an “on-rate” or “rate of association” or“association rate” or “kon” and an “off-rate” or “rate of dissociation”or “dissociation rate” or “koff” are also determined with the surfaceplasmon resonance technique using a BIAcore™-2000 or a BIAcore™-3000(BIAcore, Inc., Piscataway, N.J.). In additional embodiments, the “Kd”,“kon”, and “koff” are measured using the Octet® Systems (Pall LifeSciences).

Described herein are single domain serum albumin binding proteins,pharmaceutical compositions as well as nucleic acids, recombinantexpression vectors, and host cells for making such single domain serumalbumin binding proteins. Also provided are methods of using thedisclosed single domain serum albumin binding proteins in theprevention, and/or treatment of diseases, conditions and disorders. Thesingle domain serum albumin binding proteins are capable specificallybinding to serum albumin. In some embodiments, the single domain serumalbumin binding proteins include additional domains, such as a CD3binding domain, as well as binding domains for other target antigens.

Single Domain Serum Albumin Binding Protein

Contemplated herein are single domain serum albumin binding proteins.Serum albumin is produced by the liver, occurs dissolved in blood plasmaand is the most abundant blood protein in mammals. Albumin is essentialfor maintaining the oncotic pressure needed for proper distribution ofbody fluids between blood vessels and body tissues; without albumin, thehigh pressure in the blood vessels would force more fluids out into thetissues. It also acts as a plasma carrier by non-specifically bindingseveral hydrophobic steroid hormones and as a transport protein forhemin and fatty acids. Human serum albumin (HSA) (molecular mass ˜67kDa) is the most abundant protein in plasma, present at about 50 mg/ml(600 μM), and has a half-life of around 20 days in humans. HSA serves tomaintain plasma pH, contributes to colloidal blood pressure, functionsas carrier of many metabolites and fatty acids, and serves as a majordrug transport protein in plasma. In some embodiments, the single domainserum albumin binding proteins bind to HSA. In some embodiments, thesingle domain serum albumin binding proteins bind to serum albuminprotein from cynomolgus monkeys. In some embodiments, the single domainserum albumin binding proteins bind to HSA and serum albumin proteinfrom cynomolgus monkeys. In some embodiments, the single domain serumalbumin binding proteins also bind to mouse serum albumin protein. Insome embodiments, the binding affinity towards mouse serum albumin isabout 1.5-fold to about 20-fold weaker than that towards human orcynomolgus serum albumin.

Noncovalent association with albumin extends the elimination half-timeof short lived proteins. For example, a recombinant fusion of an albuminbinding domain to a Fab fragment resulted in a decrease in in vivoclearance by 25- and 58-fold and a half-life extension of 26- and37-fold when administered intravenously to mice and rabbits respectivelyas compared to the administration of the Fab fragment alone. In anotherexample, when insulin is acylated with fatty acids to promoteassociation with albumin, a protracted effect was observed when injectedsubcutaneously in rabbits or pigs. Together, these studies demonstrate alinkage between albumin binding and prolonged action/serum half-life.

In some embodiments, the single-domain serum albumin binding proteinsdescribed herein is a single domain antibody such as a heavy chainvariable domain (VH), a variable domain (VHH) of camelid derived sdAb,peptide, ligand or small molecule entity specific for serum albumin. Insome embodiments, the single-domain serum albumin binding proteinsdescribed herein is a single domain antibody such as a heavy chainvariable domain (VH), a variable domain (VHH) of camelid derived sdAb,peptide, ligand or small molecule entity specific for HSA. In someembodiments, the serum albumin binding domain of a single domain serumalbumin binding protein described herein is any domain that binds toserum albumin including but not limited to domains from a monoclonalantibody, a polyclonal antibody, a recombinant antibody, a humanantibody, a humanized antibody. In certain embodiments, the serumalbumin binding domain is a single-domain antibody. In otherembodiments, the serum albumin binding domain is a peptide. In furtherembodiments, the serum albumin binding domain is a small molecule. It iscontemplated that the single domain serum albumin binding protein isfairly small and no more than 25 kD, no more than 20 kD, no more than 15kD, or no more than 10 kD in some embodiments. In certain instances, thesingle domain serum albumin binding protein binding is 5 kD or less ifit is a peptide or small molecule entity.

In some embodiments, the single domain serum albumin binding proteindescribed herein is a half-life extension domain which provides foraltered pharmacodynamics and pharmacokinetics of the single domain serumalbumin binding protein itself. As above, the half-life extension domainextends the elimination half-time. The half-life extension domain alsoalters pharmacodynamic properties including alteration of tissuedistribution, penetration, and diffusion of the single domain serumalbumin binding protein. In some embodiments, the half-life extensiondomain provides for improved tissue (including tumor) targeting, tissuedistribution, tissue penetration, diffusion within the tissue, andenhanced efficacy as compared with a protein without a half-lifeextension domain. In one embodiment, therapeutic methods effectively andefficiently utilize a reduced amount of the single domain serum albuminbinding protein, resulting in reduced side effects, such as reducednon-tumor cell cytotoxicity.

Further, the binding affinity of the single domain serum albumin bindingprotein towards its binding target can be selected so as to target aspecific elimination half-time in a particular single domain serumalbumin binding protein. Thus, in some embodiments, the single domainserum albumin binding protein has a high binding affinity towards itsbinding target. In other embodiments, the single domain serum albuminbinding protein has a medium binding affinity towards its bindingtarget. In yet other embodiments, the single domain serum albuminbinding protein has a low or marginal binding affinity towards itsbinding target. Exemplary binding affinities include K_(D) of 10 nM orless (high), between 10 nM and 100 nM (medium), and greater than 100 nM(low). As above, binding affinities of the single domain serum albuminbinding proteins towards binding targets are determined by known methodssuch as Surface Plasmon Resonance (SPR).

In certain embodiments, the single domain serum albumin binding proteindisclosed herein binds to HSA with a human Kd (hKd). In certainembodiments, the single domain serum albumin binding protein disclosedherein binds to cynomolgus monkey serum albumin with a cyno Kd (cKd). Incertain embodiments, the single domain serum albumin binding proteindisclosed herein binds to cynomolgus monkey serum albumin with a cyno Kd(cKd) and to HSA with a human Kd (hKd). In some embodiments, the hKdranges between 1 nM and 100 nM. In some embodiments, the hKd rangesbetween 1 nM and 10 nM. In some embodiments, the cKd ranges between 1 nMand 100 nM. In some embodiments, the cKd ranges between 1 nM and 10 nM.In some embodiments, the hKd and the cKd range between about 1 nM andabout 5 nM or between about 5 nM and 10 nM. In some embodiments, thesingle domain serum albumin binding protein binds to serum albuminselected from human serum albumin, cynomolgus serum albumin, and mouseserum albumin. In some embodiments, the single domain serum albuminbinding protein binds to human serum albumin, cynomolgus serum albumin,and mouse serum albumin with comparable binding affinity (Kd). In someembodiments, the single domain serum albumin binding protein binds tohuman serum albumin with a human Kd (hKd) between about 1 nM and about10 nM and to cynomolgus serum albumin with a cynomolgus Kd (cKd) between1 nM and 10 nM. In some embodiments, the single domain serum albuminbinding protein binds to mouse serum albumin with a mouse Kd (mKd)between about 10 nM and about 50 nM.

In some embodiments, the hKd is about 1.5 nM, about 1.6 nM, about 1.7nM, about 1.8 nM, about 1.9 nM, about 2 nM, about 2.1 nM, about 2.2 nM,about 2.3 nM, about 2.4 nM, about 2.5 nM, about 2.6 nM, about 2.7 nM,about 2.8 nM, about 2.9 nM, about 3 nM, 3.1 nM, about 3.2 nM, about 3.3.nM, about 3.4 nM, about 3.5 nM, about 3.6 nM, about 3.7 nM, about 3.8nM, about 3.9 nM, about 4 nM, about 4.5 nM, about 5 nM, about 6, about6.5 nM, about 7 nM, about 7.5 nM, about 8 nM, about 8.5 nM, about 9.0nM, about 9.5 nM, or about 10 nM.

In some embodiments, the cKd is about 1.5 nM, about 1.6 nM, about 1.7nM, about 1.8 nM, about 1.9 nM, about 2 nM, about 2.1 nM, about 2.2 nM,about 2.3 nM, about 2.4 nM, about 2.5 nM, about 2.6 nM, about 2.7 nM,about 2.8 nM, about 2.9 nM, about 3 nM, 3.1 nM, about 3.2 nM, about 3.3.nM, about 3.4 nM, about 3.5 nM, about 3.6 nM, about 3.7 nM, about 3.8nM, about 3.9 nM, about 4 nM, about 4.5 nM, about 5 nM, about 6, about6.5 nM, about 7 nM, about 7.5 nM, about 8 nM, about 8.5 nM, about 9.0nM, about 9.5 nM, or about 10 nM.

In some embodiments, the mKd is about 10 nM, about 11 nM, about 12 nM,about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about18 nM, about 19 nM, about 20 nM, about 21 nM, about 22 nM, about 23 nM,about 24 nM, about 25 nM, about 26 nM, about 27. nM, about 28 nM, about29 nM, about 30 nM, about 31 nM, about 32 nM, about 33 nM, about 34 nM,about 35 nM, about 36 nM, about 37, about 38 nM, about 39 nM, about 40nM, about 41 nM, about 42 nM, about 43 nM, about 44 nM, about 45 nM,about 46 nM, about 47 nM, about 48 nM, or about 50 nM.

In some embodiments, the single domain serum albumin binding protein hasan amino acid sequence selected from SEQ ID NO. 4, SEQ ID NO. 5, SEQ IDNO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 25, SEQ IDNO. 26, and SEQ ID NO. 27.

In some embodiments, the single domain serum albumin binding protein hasthe amino acid sequence set forth as SEQ ID NO. 4, and the hKd and thecKd are between about 1 nM and about 5 nM. In some embodiments, thesingle domain serum albumin binding protein has the amino acid sequenceset forth as SEQ ID NO. 4, and the hKd is about 2.3 nM and the cKd isabout 2.4 nM. In some embodiments, the single domain serum albuminbinding protein has the amino acid sequence set forth as SEQ ID NO. 25,and the hKd and the cKd are between about 1 nM and about 5 nM. In someembodiments, the single domain serum albumin binding protein has theamino acid sequence set forth as SEQ ID NO. 25, and the hKd is about 2.1nM and the cKd is about 2.2 nM. In some embodiments, the single domainserum albumin binding protein has the amino acid sequence set forth asSEQ ID NO. 5, and the hKd and the cKd are between about 1 nM and about 5nM. In some embodiments, the single domain serum albumin binding proteinhas the amino acid sequence set forth as SEQ ID NO. 5, and the hKd isabout 1.9 nM and the cKd is about 1.7 nM. In some embodiments, thesingle domain serum albumin binding protein has the amino acid sequenceset forth as SEQ ID NO. 6, and the hKd and the cKd are between about 1nM and about 5 nM. In some embodiments, the single domain serum albuminbinding protein has the amino acid sequence set forth as SEQ ID NO. 6,and the hKd is about 3.2 nM and the cKd is about 3.6 nM. In someembodiments, the single domain serum albumin binding protein has theamino acid sequence set forth as SEQ ID NO. 7, and the hKd and the cKdare between about 1 nM and about 5 nM. In some embodiments, the singledomain serum albumin binding protein has the amino acid sequence setforth as SEQ ID NO. 7, and the hKd is about 2.7 nM and the cKd is about2.6 nM. In some embodiments, the single domain serum albumin bindingprotein has the amino acid sequence set forth as SEQ ID NO. 26, and thehKd and the cKd are between about 1 nM and about 5 nM. In someembodiments, the single domain serum albumin binding protein has theamino acid sequence set forth as SEQ ID NO. 26, and the hKd is about 2.1nM and the cKd is about 2 nM. In some embodiments, the single domainserum albumin binding protein has the amino acid sequence set forth asSEQ ID NO. 8, and the hKd and the cKd are between about 5 nM and about10 nM. In some embodiments, the single domain serum albumin bindingprotein has the amino acid sequence set forth as SEQ ID NO. 8, and thehKd is about 6 nM and the cKd is about 7.5 nM. In some embodiments, thesingle domain serum albumin binding protein has the amino acid sequenceset forth as SEQ ID NO. 9, and wherein the hKd and the cKd are betweenabout 1 nM and about 5 nM. In some embodiments, the single domain serumalbumin binding protein has the amino acid sequence set forth as SEQ IDNO. 9, and wherein the hKd is about 2.2 nM and the cKd is about 2.3 nM.In some embodiments, the single domain serum albumin binding protein hasthe amino acid sequence set forth as SEQ ID NO. 27 and wherein the hKdand the cKd are between about 1 nM and about 5 nM. In some embodiments,the single domain serum albumin binding protein has the amino acidsequence set forth as SEQ ID NO. 27 and wherein the hKd is about 1.6 nMand the cKd is about 1.6 nM.

In some embodiments, the single domain serum albumin binding protein hasthe amino acid sequence set forth as SEQ ID NO. 4 and has a mKd of about17 nM. In some embodiments, the single domain serum albumin bindingprotein has the amino acid sequence set forth as SEQ ID NO. 5 and has amKd of about 12 nM. In some embodiments, the single domain serum albuminbinding protein has the amino acid sequence set forth as SEQ ID NO. 6and has a mKd of about 33 nM. In some embodiments, the single domainserum albumin binding protein has the amino acid sequence set forth asSEQ ID NO. 7 and has a mKd of about 14 nM. In some embodiments, thesingle domain serum albumin binding protein has the amino acid sequenceset forth as SEQ ID NO. 9 and has a mKd of about 16 nM. In someembodiments, the single domain serum albumin binding protein has theamino acid sequence set forth as SEQ ID NO. 25 and has a mKd of about 17nM. In some embodiments, the single domain serum albumin binding proteinhas the amino acid sequence set forth as SEQ ID NO. 26 and has a mKd ofabout 17 nM. In some embodiments, the single domain serum albuminbinding protein has the amino acid sequence set forth as SEQ ID NO. 27and has a mKd of about 16 nM.

In some embodiments, the ratio between the hKd and cKd (hKd:cKd) rangesfrom about 20:1 to about 1:2.

In some embodiments, the single domain serum albumin binding protein hasan elimination half-time of at least 1 hour, at least 2 hours, at least4 hours, at least 6 hours, at least 12 hours, at least 20 hours, atleast 25 hours, at least 30 hours, at least 35 hours, at least 40 hours,at least 45 hours, at least 50 hours, or at least 100 hours.

CD3 Binding Domain

The specificity of the response of T cells is mediated by therecognition of antigen (displayed in context of a majorhistocompatibility complex, WIC) by the T cell receptor complex. As partof the T cell receptor complex, CD3 is a protein complex that includes aCD3γ (gamma) chain, a CD3δ (delta) chain, and two CD3ε (epsilon) chainswhich are present on the cell surface. CD3 associates with the α (alpha)and β (beta) chains of the T cell receptor (TCR) as well as and CD3 ζ(zeta) altogether to comprise the T cell receptor complex. Clustering ofCD3 on T cells, such as by immobilized anti-CD3 antibodies leads to Tcell activation similar to the engagement of the T cell receptor butindependent of its clone-typical specificity.

In one aspect is described herein a multispecific protein comprising asingle domain serum albumin binding protein according to the presentdisclosure. In some embodiments, the multispecific protein furthercomprises a domain which specifically binds to CD3. In some embodiments,the multispecific protein further comprises a domain which specificallybinds to human CD3. In some embodiments, the multispecific proteinfurther comprises a domain which specifically binds to CD3γ. In someembodiments, the multispecific protein further comprises a domain whichspecifically binds to CD3δ. In some embodiments, the multispecificprotein further comprises a domain which specifically binds to CD3ε.

In additional embodiments, the multispecific protein further comprises adomain which specifically binds to the T cell receptor (TCR). In someembodiments, the multispecific protein further comprises a domain whichspecifically binds the α chain of the TCR. In some embodiments, themultispecific protein further comprises a domain which specificallybinds the β chain of the TCR.

In certain embodiments, the CD3 binding domain of the multispecificprotein comprising a single domain serum albumin binding proteindescribed herein exhibit not only potent CD3 binding affinities withhuman CD3, but show also excellent crossreactivity with the respectivecynomolgus monkey CD3 proteins. In some instances, the CD3 bindingdomain of the multispecific proteins are cross-reactive with CD3 fromcynomolgus monkey. In certain instances, human:cynomolgous K_(D)(hKd:cKd) ratios for CD3 binding are between 20:1 and 1:2.

In some embodiments, the CD3 binding domain of the multispecific proteincomprising a single domain serum albumin binding protein describedherein can be any domain that binds to CD3 including but not limited todomains from a monoclonal antibody, a polyclonal antibody, a recombinantantibody, a human antibody, a humanized antibody, or antigen bindingfragments of the CD3 binding antibodies, such as single domainantibodies (sdAb), Fab, Fab′, F(ab)2, and Fv fragments, fragmentscomprised of one or more CDRs, single-chain antibodies (e.g., singlechain Fv fragments (scFv)), disulfide stabilized (dsFv) Fv fragments,heteroconjugate antibodies (e.g., bispecific antibodies), pFv fragments,heavy chain monomers or dimers, light chain monomers or dimers, anddimers consisting of one heavy chain and one light chain. In someinstances, it is beneficial for the CD3 binding domain to be derivedfrom the same species in which the multispecific protein comprising asingle domain serum albumin binding protein described herein willultimately be used in. For example, for use in humans, it may bebeneficial for the CD3 binding domain of the multispecific proteincomprising a single domain serum albumin binding protein describedherein to comprise human or humanized residues from the antigen bindingdomain of an antibody or antibody fragment.

Thus, in one aspect, the antigen-binding domain comprises a humanized orhuman antibody or an antibody fragment, or a murine antibody or antibodyfragment. In one embodiment, the humanized or human anti-CD3 bindingdomain comprises one or more (e.g., all three) light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) of a humanized or human anti-CD3 binding domain describedherein, and/or one or more (e.g., all three) heavy chain complementarydetermining region 1 (HC CDR1), heavy chain complementary determiningregion 2 (HC CDR2), and heavy chain complementary determining region 3(HC CDR3) of a humanized or human anti-CD3 binding domain describedherein, e.g., a humanized or human anti-CD3 binding domain comprisingone or more, e.g., all three, LC CDRs and one or more, e.g., all three,HC CDRs.

In some embodiments, the humanized or human anti-CD3 binding domaincomprises a humanized or human light chain variable region specific toCD3 where the light chain variable region specific to CD3 compriseshuman or non-human light chain CDRs in a human light chain frameworkregion. In certain instances, the light chain framework region is a λ(lambda) light chain framework. In other instances, the light chainframework region is a κ (kappa) light chain framework.

In some embodiments, the humanized or human anti-CD3 binding domaincomprises a humanized or human heavy chain variable region specific toCD3 where the heavy chain variable region specific to CD3 compriseshuman or non-human heavy chain CDRs in a human heavy chain frameworkregion.

In certain instances, the complementary determining regions of the heavychain and/or the light chain are derived from known anti-CD3 antibodies,such as, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4),teplizumab (MGA031), visilizumab (Nuvion), SP34, TR-66 or X35-3, VIT3,BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2,TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6,T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1 and WT-31.

The affinity to bind to CD3 can be determined, for example, by theability of the multispecific protein comprising a single domain serumalbumin binding protein itself or its CD3 binding domain to bind to CD3coated on an assay plate; displayed on a microbial cell surface; insolution; etc. The binding activity of multispecific protein comprisinga single domain serum albumin binding protein itself or its CD3 bindingdomain according to the present disclosure to CD3 can be assayed byimmobilizing the ligand (e.g., CD3) or said multispecific protein itselfor its CD3 binding domain, to a bead, substrate, cell, etc. Agents canbe added in an appropriate buffer and the binding partners incubated fora period of time at a given temperature. After washes to remove unboundmaterial, the bound protein can be released with, for example, SDS,buffers with a high pH, and the like and analyzed, for example, bySurface Plasmon Resonance (SPR).

Target Antigen Binding Domain

In addition to the described serum albumin binding and CD3 domains, themultispecific binding protein comprising a single domain serum albuminbinding proteins described herein, in certain embodiments, also comprisea domain that binds to a target antigen. A target antigen is involved inand/or associated with a disease, disorder or condition. In particular,a target antigen associated with a proliferative disease, a tumorousdisease, an inflammatory disease, an immunological disorder, anautoimmune disease, an infectious disease, a viral disease, an allergicreaction, a parasitic reaction, a graft-versus-host disease or ahost-versus-graft disease. In some embodiments, the target antigen is atumor antigen expressed on a tumor cell. Alternatively in someembodiments, the target antigen is associated with a pathogen such as avirus or bacterium.

In some embodiments, the target antigen is a cell surface molecule suchas a protein, lipid or polysaccharide. In some embodiments, the targetantigen is a on a tumor cell, virally infected cell, bacteriallyinfected cell, damaged red blood cell, arterial plaque cell, or fibrotictissue cell.

The design of the multispecific binding proteins comprising a singledomain serum albumin binding protein according to the present disclosureallows the binding domain to a target antigen to be flexible in that thebinding domain to a target antigen can be any type of binding domain,including but not limited to, domains from a monoclonal antibody, apolyclonal antibody, a recombinant antibody, a human antibody, ahumanized antibody. In some embodiments, the binding domain to a targetantigen is a single chain variable fragments (scFv), single-domainantibody such as a heavy chain variable domain (VH), a light chainvariable domain (VL) and a variable domain (VHH) of camelid derivedsdAb. In other embodiments, the binding domain to a target antigen is anon-Ig binding domain, i.e., antibody mimetic, such as anticalins,affilins, affibody molecules, affimers, affitins, alphabodies, avimers,DARPins, fynomers, kunitz domain peptides, and monobodies. In furtherembodiments, the binding domain to a target antigen is a ligand orpeptide that binds to or associates with a target antigen. In yetfurther embodiments, the binding domain to a target antigen is aknottin. In yet further embodiments, the binding domain to a targetantigen is a small molecular entity.

Single Domain Serum Albumin Binding Protein Modifications

The single domain serum albumin binding proteins described hereinencompass derivatives or analogs in which (i) an amino acid issubstituted with an amino acid residue that is not one encoded by thegenetic code, (ii) the mature polypeptide is fused with another compoundsuch as polyethylene glycol, or (iii) additional amino acids are fusedto the protein, such as a leader or secretory sequence or a sequence toblock an immunogenic domain and/or for purification of the protein.

Typical modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphatidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation, myristylation,oxidation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.

Modifications are made anywhere in single domain serum albumin bindingproteins described herein, including the peptide backbone, the aminoacid side-chains, and the amino or carboxyl termini. Certain commonpeptide modifications that are useful for modification of single domainserum albumin binding proteins include glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation,blockage of the amino or carboxyl group in a polypeptide, or both, by acovalent modification, and ADP-ribosylation.

Polynucleotides Encoding Single Domain Serum Albumin Binding Proteins

Also provided, in some embodiments, are polynucleotide moleculesencoding a single domain serum albumin binding protein described herein.In some embodiments, the polynucleotide molecules are provided as a DNAconstruct. In other embodiments, the polynucleotide molecules areprovided as a messenger RNA transcript.

The polynucleotide molecules are constructed by known methods such as bycombining the genes encoding the three binding domains either separatedby peptide linkers or, in other embodiments, directly linked by apeptide bond, into a single genetic construct operably linked to asuitable promoter, and optionally a suitable transcription terminator,and expressing it in bacteria or other appropriate expression systemsuch as, for example CHO cells.

Also provided, in some embodiments, are polynucleotide moleculesencoding a multispecific binding protein comprising a single domainserum albumin binding protein according to the present disclosure. Insome embodiments, the polynucleotide encoding said multispecific bindingprotein also includes coding sequence for a CD3 binding domain. In someembodiments, the polynucleotide encoding said multispecific bindingprotein also includes coding sequence for a target antigen bindingdomain. In some embodiments, the polynucleotide encoding saidmultispecific binding protein also includes coding sequences for a CD3binding domain and a target antigen binding domain. In some embodiments,the polynucleotide molecules are provided as a DNA construct. In otherembodiments, the polynucleotide molecules are provided as a messengerRNA transcript. In the embodiments where the target antigen bindingdomain is a small molecule, the polynucleotides contain genes encodingthe serum albumin binding domain and the CD3 binding domain. In theembodiments where the half-life extension domain is a small molecule,the polynucleotides contain genes encoding the domains that bind to CD3and the target antigen. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Thepromoter is selected such that it drives the expression of thepolynucleotide in the respective host cell.

In some embodiments, the polynucleotide is inserted into a vector,preferably an expression vector, which represents a further embodiment.This recombinant vector can be constructed according to known methods.Vectors of particular interest include plasmids, phagemids, phagederivatives, virii (e.g., retroviruses, adenoviruses, adeno-associatedviruses, herpes viruses, lentiviruses, and the like), and cosmids.

A variety of expression vector/host systems may be utilized to containand express the polynucleotide encoding the polypeptide of the describedsingle domain serum albumin binding protein. Examples of expressionvectors for expression in E. coli are pSKK (Le Gall et al., J ImmunolMethods. (2004) 285(1):111-27), pcDNA5 (Invitrogen) for expression inmammalian cells, PICHIAPINK™ Yeast Expression Systems (Invitrogen),BACUVANCE™ Baculovirus Expression System (GenScript).

Thus, the single domain serum albumin binding proteins as describedherein, in some embodiments, are produced by introducing a vectorencoding the protein as described above into a host cell and culturingsaid host cell under conditions whereby the protein domains areexpressed, may be isolated and, optionally, further purified.

Production of Single Domain Serum Albumin Binding Proteins

Disclosed herein, in some embodiments, is a process for the productionof a single domain serum albumin binding protein. In some embodiments,the process comprises culturing a host transformed or transfected with avector comprising a nucleic acid sequence encoding a single domain serumalbumin binding protein under conditions allowing the expression of theserum albumin binding protein and recovering and purifying the producedprotein from the culture.

In an additional embodiment is provided a process directed to improvingone or more properties, e.g. affinity, stability, heat tolerance,cross-reactivity, etc., of the single domain serum albumin bindingproteins and/or the multispecific binding proteins comprising a singledomain serum albumin binding protein described herein, compared to areference binding compound. In some embodiments, a plurality ofsingle-substitution libraries is provided each corresponding to adifferent domain, or amino acid segment of the single domain serumalbumin binding protein or reference binding compound such that eachmember of the single-substitution library encodes only a single aminoacid change in its corresponding domain, or amino acid segment. (Thisallows all of the potential substitutions in a large protein or proteinbinding site to be probed with a few small libraries.) In someembodiments, the plurality of domains forms or covers a contiguoussequence of amino acids of the single domain serum albumin bindingprotein or a reference binding compound. Nucleotide sequences ofdifferent single-substitution libraries overlap with the nucleotidesequences of at least one other single-substitution library. In someembodiments, a plurality of single-substitution libraries are designedso that every member overlaps every member of each single-substitutionlibrary encoding an adjacent domain.

Binding compounds expressed from such single-substitution libraries areseparately selected to obtain a subset of variants in each library whichhas properties at least as good as those of the reference bindingcompound and whose resultant library is reduced in size. (That is, thenumber of nucleic acids encoding the selected set of binding compoundsis smaller than the number of nucleic acids encoding members of theoriginal single-substitution library) Such properties include, but arenot limited to, affinity to a target compound, stability with respect tovarious conditions such as heat, high or low pH, enzymatic degradation,cross-reactivity to other proteins and the like. The selected compoundsfrom each single-substitution library are referred to hereininterchangeably as “pre-candidate compounds,” or “pre-candidateproteins.” Nucleic acid sequences encoding the pre-candidate compoundsfrom the separate single-substitution libraries are then shuffled in aPCR to generate a shuffled library, using PCR-based gene shufflingtechniques.

An exemplary work flow of the screening process is described herein.Libraries of pre-candidate compounds are generated from singlesubstitution libraries and selected for binding to the targetprotein(s), after which the pre-candidate libraries are shuffled toproduce a library of nucleic acids encoding candidate compounds which,in turn, are cloned into a convenient expression vector, such as aphagemid expression system. Phage expressing candidate compounds thenundergo one or more rounds of selection for improvements in desiredproperties, such as binding affinity to a target molecule. Targetmolecules may be adsorbed or otherwise attached to a surface of a wellor other reaction container, or target molecules may be derivatized witha binding moiety, such as biotin, which after incubation with candidatebinding compounds may be captured with a complementary moiety, such asstreptavidin, bound to beads, such as magnetic beads, for washing. Inexemplary selection regimens, the candidate binding compounds undergo aprolonged wash step so that only candidate compounds with very lowdissociation rates from a target molecule are selected. Exemplary washtimes for such embodiments are at least 8 hours; or in otherembodiments, at least 24 hours; or in other embodiments, at least 48hours; or in other embodiments, at least 72 hours. Isolated clones afterselection are amplified and subjected to an additional cycle ofselection or analyzed, for example by sequencing and by makingcomparative measurements of binding affinity, for example, by ELISA,surface plasmon resonance binding, bio-layer interferometry (e.g. Octetsystem, ForteBio, Menlo Park, Calif.) or the like. In some embodiments,the process is implemented to identify one or more a single domain serumalbumin binding proteins and/or a multispecific binding proteincomprising a single domain serum albumin binding protein with improvedthermal stability, improved cross reactivity to a selected set ofbinding targets compared to that of a reference serum albumin bindingprotein, such as a protein having the amino acid sequence of SEQ ID NO.10. Single substitution libraries are prepared by varying codons in theVH region of the reference serum albumin binding protein, including bothcodons in framework regions and in CDRs; in another embodiment, thelocations where codons are varied comprise the CDRs of the heavy chainof the reference serum albumin binding protein, or a subset of suchCDRs, such as solely CDR1, solely CDR2, solely CDR3, or pairs thereof.In another embodiment, locations where codons are varied occur solely inframework regions. In some embodiments, a library comprises single codonchanges solely from a reference serum albumin binding protein solely inframework regions of VH numbering in the range of from 10 to 250. Inanother embodiment, the locations where codons are varied comprise theCDR3s of the heavy chain of the reference serum albumin binding protein,or a subset of such CDR3s. In another embodiment, the number oflocations where codons of VH encoding regions are varied are in therange of from 10 to 250, such that up to 100 locations are in frameworkregion. After preparation of the single substitution library, asoutlined above, the following steps are carried out: (a) expressingseparately each member of each single substitution library as apre-candidate protein; (b) selecting members of each single substitutionlibrary which encode pre-candidate proteins which bind to a bindingpartner that may or may not differ from the original binding target[e.g. a desired cross-reaction target(s)]; (c) shuffling members of theselected libraries in a PCR to produce a combinatorial shuffled library;(d) expressing members of the shuffled library as candidate serumalbumin binding proteins; and (e) selecting members of the shuffledlibrary one or more times for candidate serum albumin binding proteinswhich bind the original binding partner and potentially (f) furtherselecting the candidate proteins for binding to the desiredcross-reactive target(s) thereby providing a nucleic acid encoded serumalbumin binding protein with increased cross reactivity for the one ormore substances with respect to the reference serum albumin bindingprotein without loss of affinity for the original ligand. In additionalembodiments, the method may be implemented for obtaining a serum albuminbinding protein with decreased reactivity to a selected cross-reactivesubstance(s) or compound(s) or epitope(s) by substituting step (f) withthe following step: depleting candidate binding compounds one or moretimes from the subset of candidate serum albumin binding protein whichbind to the undesired cross-reactive compound.

Recent studies have reported that during manufacturing, storage and invivo use, therapeutic antibodies are at risk for degradation via anumber of pathways. Amongst the most frequently occurring chemicaldegradation reactions in proteins are deamidation of asparagine (N) andisomerization of aspartic acid (D) residues. In particular, it has beenhypothesized that if N and D residues are involved in antigenrecognition, their chemical alteration can lead to severe loss ofpotency. Asparagine and aspartic acid residues are known to share adegradation pathway that proceeds via the formation of a cyclicsuccinimide intermediate. The formation of succinimide intermediates andtheir hydrolysis products (aspartate and isoaspartate), at the asparticacid sites of an antibody represents a stability problem. Whenisomerization occurs, the chemical structure of the antibody alters,which could lead to poor stability, manifested for example byaggregation, shorter shelf-life. Accordingly, in some embodiments of thepresent disclosure are provided single domain serum albumin bindingproteins wherein one or more aspartic acid residue is mutated, therebyreducing the isomerization potential of the single domain serum albuminbinding protein. In some embodiments, the aspartic acid residue is inCDR2 of the single domain serum albumin binding protein and the asparticacid residue is mutated to glutamic acid. In certain embodiments, theaspartic acid residue in position 62 of the protein defined by SEQ IDNO. 10 is mutated to glutamic acid (D62E). In some embodiments, theserum albumin binding affinity of the single domain serum albuminproteins containing the D62Emutation is not affected by the mutation. Insome embodiments, single domain serum albumin binding proteins with andwithout the D62E mutation have comparable binding affinity towards serumalbumin.

Pharmaceutical Compositions

Also provided, in some embodiments, are pharmaceutical compositionscomprising a single domain serum albumin binding protein describedherein, a vector comprising the polynucleotide encoding the polypeptideof the single domain serum albumin binding proteins or a host celltransformed by this vector and at least one pharmaceutically acceptablecarrier. The term “pharmaceutically acceptable carrier” includes, but isnot limited to, any carrier that does not interfere with theeffectiveness of the biological activity of the ingredients and that isnot toxic to the patient to whom it is administered. Examples ofsuitable pharmaceutical carriers are well known in the art and includephosphate buffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc. Suchcarriers can be formulated by conventional methods and can beadministered to the subject at a suitable dose. Preferably, thecompositions are sterile. These compositions may also contain adjuvantssuch as preservative, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents.

In some embodiments of the pharmaceutical compositions, the singledomain serum albumin binding protein described herein is encapsulated innanoparticles. In some embodiments, the nanoparticles are fullerenes,liquid crystals, liposome, quantum dots, superparamagneticnanoparticles, dendrimers, or nanorods. In other embodiments of thepharmaceutical compositions, the single domain serum albumin bindingprotein is attached to liposomes. In some instances, the single domainserum albumin binding protein is conjugated to the surface of liposomes.In some instances, the single domain serum albumin binding protein isencapsulated within the shell of a liposome. In some instances, theliposome is a cationic liposome.

The single domain serum albumin binding proteins described herein arecontemplated for use as a medicament. Administration is effected bydifferent ways, e.g. by intravenous, intraperitoneal, subcutaneous,intramuscular, topical or intradermal administration. In someembodiments, the route of administration depends on the kind of therapyand the kind of compound contained in the pharmaceutical composition.The dosage regimen will be determined by the attending physician andother clinical factors. Dosages for any one patient depends on manyfactors, including the patient's size, body surface area, age, sex, theparticular compound to be administered, time and route ofadministration, the kind of therapy, general health and other drugsbeing administered concurrently. An “effective dose” refers to amountsof the active ingredient that are sufficient to affect the course andthe severity of the disease, leading to the reduction or remission ofsuch pathology and may be determined using known methods.

Methods of Treatment

Also provided herein, in some embodiments, are methods and uses forstimulating the immune system of an individual in need thereofcomprising administration of a single domain serum albumin bindingprotein or a multispecific binding protein comprising the a singledomain serum albumin binding protein described herein. In someinstances, the administration of a single domain serum albumin bindingprotein described herein induces and/or sustains cytotoxicity towards acell expressing a target antigen. In some instances, the cell expressinga target antigen is a cancer or tumor cell, a virally infected cell, abacterially infected cell, an autoreactive T or B cell, damaged redblood cells, arterial plaques, or fibrotic tissue.

Also provided herein are methods and uses for a treatment of a disease,disorder or condition associated with a target antigen comprisingadministering to an individual in need thereof a single domain serumalbumin binding protein or a multispecific binding protein comprisingthe a single domain serum albumin binding protein described herein.Diseases, disorders or conditions associated with a target antigeninclude, but are not limited to, viral infection, bacterial infection,auto-immune disease, transplant rejection, atherosclerosis, or fibrosis.In other embodiments, the disease, disorder or condition associated witha target antigen is a proliferative disease, a tumorous disease, aninflammatory disease, an immunological disorder, an autoimmune disease,an infectious disease, a viral disease, an allergic reaction, aparasitic reaction, a graft-versus-host disease or a host-versus-graftdisease. In one embodiment, the disease, disorder or conditionassociated with a target antigen is cancer. In one instance, the canceris a hematological cancer. In another instance, the cancer is a solidtumor cancer.

As used herein, in some embodiments, “treatment” or “treating” or“treated” refers to therapeutic treatment wherein the object is to slow(lessen) an undesired physiological condition, disorder or disease, orto obtain beneficial or desired clinical results. For the purposesdescribed herein, beneficial or desired clinical results include, butare not limited to, alleviation of symptoms; diminishment of the extentof the condition, disorder or disease; stabilization (i.e., notworsening) of the state of the condition, disorder or disease; delay inonset or slowing of the progression of the condition, disorder ordisease; amelioration of the condition, disorder or disease state; andremission (whether partial or total), whether detectable orundetectable, or enhancement or improvement of the condition, disorderor disease. Treatment includes eliciting a clinically significantresponse without excessive levels of side effects. Treatment alsoincludes prolonging survival as compared to expected survival if notreceiving treatment. In other embodiments, “treatment” or “treating” or“treated” refers to prophylactic measures, wherein the object is todelay onset of or reduce severity of an undesired physiologicalcondition, disorder or disease, such as, for example is a person who ispredisposed to a disease (e.g., an individual who carries a geneticmarker for a disease such as breast cancer).

In some embodiments of the methods described herein, the single domainserum albumin binding proteins or a multispecific binding proteincomprising the a single domain serum albumin binding protein describedherein are administered in combination with an agent for treatment ofthe particular disease, disorder or condition. Agents include but arenot limited to, therapies involving antibodies, small molecules (e.g.,chemotherapeutics), hormones (steroidal, peptide, and the like),radiotherapies (γ-rays, X-rays, and/or the directed delivery ofradioisotopes, microwaves, UV radiation and the like), gene therapies(e.g., antisense, retroviral therapy and the like) and otherimmunotherapies. In some embodiments, the single domain serum albuminbinding proteins or a multispecific binding protein comprising the asingle domain serum albumin binding protein described herein areadministered in combination with anti-diarrheal agents, anti-emeticagents, analgesics, opioids and/or non-steroidal anti-inflammatoryagents. In some embodiments, the single domain serum albumin bindingproteins or a multispecific binding protein comprising a single domainserum albumin binding protein as described herein are administeredbefore, during, or after surgery.

EXAMPLES Example 1: Generation of Anti-HSA Single Domain AntibodyVariants with Equivalent Binding Properties to a Parental Anti-HSASingle Domain Antibody Characterization of Parental Anti-HSA Phage

Specific binding of the parental anti-HSA phage to an HSA antigen wasdetermined, using CD3 as a negative control (FIG. 1) and crossreactivity of the anti-HSA phage to human, cynomolgus monkey, and mouseserum albumin was determined (FIG. 2).

Single Substitution HSA sdAb Phage Libraries

A single substitution library was provided for each of the three CDRdomains. Single substitution libraries were bound to HSA and then washedin buffer containing various levels of HSA. Phages bound at 0 and 24hours were rescued and counted. Phages selected using a 24 hour washwith 2.5 mg/ml HSA in the buffer were used to create two independentcombinatorial phage libraries.

Combinatorial Anti-HSA Libraries

MSA was used as the selection target in the first Round. Wells werewashed for 24 hours after combinatorial phage binding from twoindependent libraries. HSA was used as the selection target in thesecond round. Wells were washed in 1 mg/ml HSA for 24 hours afterbinding of both libraries. Inserts PCRed from the second round ofselection were subcloned into the ME10 His6 expression vector (6×Hissequence disclosed as SEQ ID NO: 38). 96 clones were picked, DNA waspurified, sequenced, and transfected into Expi293 cells.

Binding Affinity Measurement

Supernatants were used to estimate Kd to HSA and CSA using the Octetplatform. Nine clones were selected for further characterization (FIG.3), based on binding affinities compared to the parental sdAb as well asrobust production, aggregation and stability profiles.

Example 2: Pharmacokinetics of a Trispecific Antibody Comprising theAnti-HSA Single Domain Antibody

The anti-HSA single domain antibody of Example 1 is used to prepare atrispecific antibody which is evaluated for half-time elimination inanimal studies.

The trispecific antibody is administered to cynomolgus monkeys as a 0.5mg/kg bolus injection intramuscularly. Another cynomolgus monkey groupreceives a comparable protein in size with binding domains to CD3 andCD20, but lacking HSA binding. A third and fourth group receive anantibody with CD3 and HSA binding domains and a protein with CD20 andHSA binding domains respectively, and both comparable in size to thetrispecific antibody. Each test group consists of 5 monkeys. Serumsamples are taken at indicated time points, serially diluted, and theconcentration of the proteins is determined using a binding ELISA to CD3and/or CD20.

Pharmacokinetic analysis is performed using the test article plasmaconcentrations. Group mean plasma data for each test article conforms toa multi-exponential profile when plotted against the time post-dosing.The data are fit by a standard two-compartment model with bolus inputand first-order rate constants for distribution and elimination phases.The general equation for the best fit of the data for i.v.administration is: c(t)=Ae^(−αt)+Be^(−βt), where c(t) is the plasmaconcentration at time t, A and B are intercepts on the Y-axis, and α andβ are the apparent first-order rate constants for the distribution andelimination phases, respectively. The α-phase is the initial phase ofthe clearance and reflects distribution of the protein into allextracellular fluid of the animal, whereas the second or β-phase portionof the decay curve represents true plasma clearance. Methods for fittingsuch equations are well known in the art. For example,A=D/V(α−k21)/(α−β), B=D/V(β−k21)/(α−β), and α and β (for α>β) are rootsof the quadratic equation: r²+(k12+k21+k10)r+k21k10=0 using estimatedparameters of V=volume of distribution, k10=elimination rate,k12=transfer rate from compartment 1 to compartment 2 and k21=transferrate from compartment 2 to compartment 1, and D=the administered dose.

Data analysis: Graphs of concentration versus time profiles are madeusing KaleidaGraph (KaleidaGraph™ V. 3.09 Copyright 1986-1997. SynergySoftware. Reading, Pa.). Values reported as less than reportable (LTR)are not included in the PK analysis and are not represented graphically.Pharmacokinetic parameters are determined by compartmental analysisusing WinNonlin software (WinNonlin® Professional V. 3.1 WinNonlin™Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.).Pharmacokinetic parameters are computed as described in Ritschel W A andKearns G L, 1999, IN: Handbook Of Basic Pharmacokinetics IncludingClinical Applications, 5th edition, American Pharmaceutical Assoc.,Washington, D.C.

It is expected that the trispecific antibody comprising the anti-HSAsingle domain antibody of Example 1 has improved pharmacokineticparameters such as an increase in elimination half-time as compared toproteins lacking an HSA binding domain.

Example 3: Thermal Stability of Anti-HSA Single Domain Antibody Variants

The temperature of hydrophobic exposure (T_(h)) of a protein correspondsto the derivative of the inflection point of peak dye fluorescence andis known to correlate with melting temperature (T_(m)), which is ameasure of protein stability. The goal of this study was to assess theT_(h) for several anti-HAS single domain antibody variants.

Protein Production

Sequences of anti-huALB single domain antibodies were cloned intopcDNA3.4 (Invitrogen) preceded by a leader sequence and followed by a 6×Histidine tag (SEQ ID NO: 38). Expi293F cells (Life Technologies A14527)were maintained in suspension in Optimum Growth Flasks (Thomson) between0.2 to 8×1e6 cells/ml in Expi 293 media. Purified plasmid DNA wastransfected into Expi293F cells in accordance with Expi293 ExpressionSystem Kit (Life Technologies, A14635) protocols, and maintained for 4-6days post transfection. Conditioned media was partially purified byaffinity and desalting chromatography. Anti-huCD3e scFv proteins wereconcentrated with Amicon Ultra centrifugal filtration units (EMDMillipore), applied to Superdex 200 size exclusion media (GE Healthcare)and resolved in a neutral buffer containing excipients. Fraction poolingand final purity were assessed by SDS-PAGE and analytical size exclusionchromatography (SEC). The absorbance of purified protein solutions weredetermined at 280 nm using a SpectraMax M2 (Molecular Devices) andUV-transparent 96-well plates (Corning 3635) and their concentrationswere calculated from molar extinction coefficients.

Differential Scanning Fluorimetry

Purified anti-HSA single domain antibody proteins were diluted from 0.2to 0.25 mg/ml together with 5×SYPRO orange dye (Life Technologies 56651)in 0.15% DMSO final concentration in a neutral buffer containingexcipients into MicroAmp EnduraPlate optical microplates and adhesivefilm (Applied Biosystems 4483485 and 4311971). A plate containingdiluted protein and dye mixtures was loaded into an ABI 7500 Fastreal-time PCR instrument (Applied Biosytems) and subjected to amulti-step thermal gradient from 25° C. to 95° C. The thermal gradientcomprised of a two minute hold at each one degree Celsius withexcitation at 500 nm and emission collected with a ROX filter. T_(h) indegrees celcius is presented for purified anti-HSA single domainantibody proteins in FIG. 4.

Example 4: Relative Propensity of Dimerization of Anti-HSA Single DomainAntibody Variants when Exposed to Low pH

Anti-HSA single domain antibody proteins were expressed in Expi293-Fcells as above. Conditioned medium for each variant was applied toprotein A agarose (GE Healthcare, 17519901) packed in a column, washedextensively with TRIS buffered saline, eluted with 0.05% (vol/vol)acetic acid at pH 3, held at room temperature for up to ten minutesprior to partial neutralization to pH 5, and subsequently desalted intoa neutral buffer containing excipients using Sephadex G25 columns (GEHealthcare 17058401).

Concentrations of purified anti-HSA single domain antibody variants weredetermined by absorbance at 280 nm as described in Example 3. Purifiedproteins were evaluated by SDS-PAGE and analytical SEC using a Yarra2000 SEC column (Phenomenex 00H-4512-E0) resolved in phosphate buffercontaining solvent on a 1200 LC with Chemstation software (Agilent).Peaks corresponding to dimer and monomer were manually integrated andvalues are presented in FIG. 5.

SEQ ID NO: Description AA Sequence  1 CDR1 with variant positionsGFX₁X₂X₃X₄FGMS  2 CDR2 with variant positions SISGSGX₅X₆TLYAX₇SX₈K  3CDR3 with variant positions GGSLX₉X₁₀  4 Anti-HSA sdAb clone 6CEVQLVESGGGLVQPGNSLRLSCAASGFTFSRFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSS 5 Anti-HSA sdAb clone 7A EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGADTLYADSLKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSKSSQGTLVTVSS 6 Anti-HSA sdAb clone 7G EVQLVESGGGLVQPGNSLRLSCAASGFTYSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSKSSQGTLVTVSS 7 Anti-HSA sdAb clone 8H EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGTDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVTVSS 8 Anti-HSA sdAb clone 9A EVQLVESGGGLVQPGNSLRLSCAASGFTFSRFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSKSSQGTLVTVSS 9 Anti-HSA sdAb clone 10G EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSVSSQGTLVTVSS10 wt anti-HSA EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSS11 wt anti-HSA CDR1 GFTFSSFGMS 12 wt anti-HSA CDR2 SISGSGSDTLYADSVK 13wt anti-HSACDR3 GGSLSR 14 CDR1 variant 1 GFTFSRFGMS 15 CDR1 variant 2GFTFSKFGMS 16 CDR1 variant 3 GFTYSSFGMS 17 CDR2 variant 1SISGSGADTLYADSLK 18 CDR2 variant 2 SISGSGTDTLYADSVK 19 CDR2 variant 3SISGSGRDTLYADSVK 20 CDR2 variant 4 SISGSGSDTLYAESVK 21 CDR2 variant 5SISGSGTDTLYAESVK 22 CDR2 variant 6 SISGSGRDTLYAESVK 23 CDR3 variant 1GGSLSK 24 CDR3 variant 2 GGSLSV 25 Anti-HSA sdAb clone 6CEEVQLVESGGGLVQPGNSLRLSCAASGFTFSRFGMSWVRQAPGKGLEWVSSISGSGSDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSS26 Anti-HSA sdAb clone 8HE EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGTDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSS27 Anti-HSA sdAb clone 10GEEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSVSSQGTLVTVSS28 Exemplary linker sequence (GS)n 29 Exemplary linker sequence (GGS)n30 Exemplary linker sequence (GGGS)n 31 Exemplary linker sequence(GGSG)n 32 Exemplary linker sequence (GGSGG)n 33Exemplary linker sequence (GGGGS)n 34 Exemplary linker sequence (GGGGG)n35 Exemplary linker sequence (GGG)n 36 Exemplary linker sequence(GGGGS)3 37 Exemplary linker sequence (GGGGS)4 38 6X Histidine HHHHHH

What is claimed is:
 1. A single domain serum albumin binding protein,comprising complementarity determining regions CDR1, CDR2, and CDR3,wherein (a) the amino acid sequence of CDR1 is as set forth inGFX₁X₂X₃X₄FGMS (SEQ ID NO. 1), X₁ is threonine, arginine, lysine,serine, or proline, X₂ is phenylalanine or tyrosine, X₃ is serine,arginine or lysine, X₄ is serine, lysine, arginine, or alanine; (b) theamino acid sequence of CDR2 is as set forth in SISGSGX₅X₆TLYAX₇SX₈K (SEQID NO. 2), X₅ is serine, arginine, threonine, or alanine, X₆ is asparticacid, histidine, valine, or threonine, X₇ is aspartic acid, histidine,arginine, or serine, X₈ is valine or leucine; and (c) the amino acidsequence of CDR3 is as set forth in GGSLX₉X₁₀ (SEQ ID NO. 3), X₉ isserine, arginine, threonine, or lysine, and X₁₀ is arginine, lysine,valine, proline, or asparagine, wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈,X₉, and X₁₀ are not simultaneously threonine, phenylalanine, serine,serine, serine, aspartic acid, aspartic acid, valine, serine, andarginine, respectively.
 2. The single domain serum albumin bindingprotein of claim 1, wherein said protein comprises the followingformula:f1-r1-f2-r2-f3-r3-f4 wherein, r1 is SEQ ID NO. 1; r2 is SEQ ID NO. 2;and r3 is SEQ ID NO. 3; and wherein f₁, f₂, f₃ and f₄ are frameworkresidues selected so that said protein is at least eighty percentidentical to the amino acid sequence set forth in SEQ ID NO.
 10. 3. Thesingle domain serum albumin binding protein of claim 2, wherein r1comprises SEQ ID NO. 14, SEQ ID NO. 15 or SEQ ID NO.
 16. 4. The singledomain serum albumin binding protein of claim 2, wherein r2 comprisesSEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO.21, or SEQ ID NO.
 22. 5. The single domain serum albumin binding proteinof claim 2, wherein r3 comprises SEQ ID NO. 23 or SEQ ID NO.
 24. 6. Thesingle domain serum albumin binding protein of claim 2, wherein r1comprises SEQ ID NO.
 14. 7. The single domain serum albumin bindingprotein of claim 2, wherein r1 comprises SEQ ID NO. 15, r2 is SEQ ID NO.17, and r3 is SEQ ID NO.
 23. 8. The single domain serum albumin bindingprotein of claim 2, wherein r1 comprises SEQ ID NO. 16, r3 comprises SEQID NO.
 23. 9. The single domain serum albumin binding protein of claim2, wherein r1 comprises SEQ ID NO. 15 and r2 comprises SEQ ID NO. 18.10. The single domain serum albumin binding protein of claim 2, whereinr1 comprises SEQ ID NO. 14 and r3 comprises SEQ ID NO.
 23. 11. Thesingle domain serum albumin binding protein of claim 2, wherein r1comprises SEQ ID NO. 15, r2 comprises SEQ ID NO. 19 and r3 comprises SEQID NO.
 24. 12. The single domain serum albumin binding protein of claim2, wherein r1 comprises SEQ ID NO. 14 and r2 comprises SEQ ID NO. 20.13. The single domain serum albumin binding protein of claim 2, whereinr1 comprises SEQ ID NO. 15, and r2 comprises SEQ ID NO.
 21. 14. Thesingle domain serum albumin binding protein of claim 2, wherein r1comprises SEQ ID NO. 15, r2 comprises SEQ ID NO. 22 and r3 comprises SEQID NO.
 24. 15. The single domain serum albumin binding protein of claim1, wherein said protein has an amino acid sequence selected from SEQ IDNO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ IDNO. 9, SEQ IN NO. 25, SEQ ID NO. 26, and SEQ ID NO.
 27. 16. The singledomain serum albumin binding protein of claim 15, wherein said proteincomprises the amino acid sequence set forth as SEQ ID NO.
 4. 17. Thesingle domain serum albumin binding protein of claim 15, wherein saidprotein comprises the amino acid sequence set forth as SEQ ID NO.
 7. 18.The single domain serum albumin binding protein of claim 15, whereinsaid protein comprises the amino acid sequence set forth as SEQ ID NO.9.
 19. The single domain serum albumin binding protein of claim 15,wherein said protein comprises the amino acid sequence set forth as SEQID NO.
 26. 20. The single domain serum albumin binding protein of claim15, wherein said protein comprises the amino acid sequence set forth asSEQ ID NO.
 27. 21. The single domain serum albumin binding protein ofclaim 1, wherein said protein binds to mouse serum albumin with abinding affinity (Kd) that is about 1.5 fold to about 20 fold weakerthan the binding affinity (Kd) of said protein towards human andcynomolgus serum albumin.
 22. The single domain serum albumin bindingprotein of claim 21, wherein said protein binds to human serum albuminwith a human Kd (hKd), to cynomolgus serum albumin with a cynomolgus Kd(cKd), and wherein ratio between the hKd and cKd (hKd:cKd) ranges fromabout 20:1 to about 1:2.
 23. The domain serum albumin binding protein ofclaim 1, wherein said protein has an elimination half-time of at least12 hours, at least 20 hours, at least 25 hours, at least 30 hours, atleast 35 hours, at least 40 hours, at least 45 hours, at least 50 hours,or at least 100 hours.
 24. A single domain serum albumin binding proteincomprising CDR1, CDR2, and CDR3, comprising the sequence set forth asSEQ ID NO. 10 (wt anti-HSA) wherein one or more amino acid residuesselected from amino acid positions 28, 29, 30, or 31 of CDR1; positions56, 57, 62, or 64 of CDR2; and positions 103, and 104 of CDR3 aresubstituted, wherein amino acid position 28 is substituted witharginine, lysine, serine, or proline, amino acid position 29 issubstituted with tyrosine, amino acid position 30 is substituted witharginine or lysine, amino acid position 31 is substituted with lysine,arginine, or alanine, amino acid position 56 is substituted witharginine, threonine, or alanine, amino acid position 57 is substitutedwith histidine, valine, or threonine, amino acid position 62 issubstituted with histidine, arginine, glutamic acid, or serine, aminoacid position 64 is substituted with leucine, amino acid position 103 issubstituted with arginine, threonine, or lysine, amino acid position 104is substituted with lysine, valine, proline, or asparagine.
 25. Thesingle domain serum albumin binding protein of claim 24, comprising oneor more additional substitutions in amino acid positions other thanpositions 28, 29, 30, 31, 56, 57, 62, 64, 103, and
 104. 26. A singledomain serum albumin binding protein comprising at least one mutation inCDR1, CDR2 or CDR3, wherein CDR1 has the sequence as set forth is SEQ IDNO:11, CDR2 has the sequence as set forth in SEQ ID NO:12, CDR3 has thesequence as set forth in SEQ ID NO. 13, and wherein the at least onemutation is not in amino acid positions 1, 2, 7, 8, 9, or 10 of SEQ IDNO: 11, positions 1, 3, 6, 10, or 11 of SEQ ID NO: 12, or positions 1 or2 of SEQ ID NO:
 13. 27. The single domain serum albumin binding proteinof claim 26, comprising at least one mutation in amino acid positionsselected from positions 3, 4, 5, and 6 of CDR1 (SEQ ID NO: 11), aminoacid positions 7, 8, 13, and 15 of CDR2 (SEQ ID NO: 12), and amino acidpositions 5 and 6 of CDR3 (SEQ ID NO: 13).
 28. The single domain serumalbumin binding protein of claim 27, comprising one or more additionalsubstitutions in amino acid positions other than 3, 4, 5, and 6 of CDR1(SEQ ID NO: 11), amino acid positions 7, 8, 13, and 15 of CDR2 (SEQ IDNO: 12), and amino acid positions 5 and 6 of CDR3 (SEQ ID NO: 13).
 29. Amethod for the treatment or amelioration of a proliferative disease, atumorous disease, an inflammatory disease, an immunological disorder, anautoimmune disease, an infectious disease, a viral disease, an allergicreaction, a parasitic reaction, a graft-versus-host disease or ahost-versus-graft disease comprising the administration of the singledomain serum albumin binding protein according to claim 1, to a subjectin need thereof.
 30. A multispecific binding protein comprising thesingle domain serum albumin binding protein according to claim 1.